Re: Tyramide amplification

On Sat, 13 May 2000, Greg Dobbin wrote:
> Could someone provide me with a brief explanation of the tyramide
> amplification technique and perhaps a reference or two, that would
> describe the method.
The terms "tyramide amplification" and "CARD" (catalysed
reporter deposition" are a little confusing. The method is
actually a sensitive technique for detecting peroxidase
activity, as in the case of horseradish peroxidase
deposited in a section as a result of immunohistochemistry
or in situ hybridization. It has been most used with
fluorescent in sit. hyb. (FISH).
For example, you can make a biotin-tyramine conjugate
by reacting tyramine with a succinimide derivative of
biotin. The tyramine end of the new molecule acts
rather like the chromogen in an ordinary peroxidase
method. In the presence of hydrogen peroxide, it is
oxidized at the sites of peroxidase activity. The
product of oxidation is unstable (possibly a free
radical; chemistry is not fully worked out) and it
covalently binds, immediately, to the section (possibly
to tyrosine side-chains of protein). One molecule of
HRP can catalyse the oxidation of many molecules of
biotin-tyramine, so tissue-bound biotin piles up at
sites of enzyme activity. It's covalently bound, so
it doesn't wash off - even if you remove the original
antibodies with acid washes. The bound biotin can be
detected with any form of labelled avidin or streptavidin:
for example, a fluorescently labelled avidin or an
HRP-labelled avidin. (A 2nd HRP label would be detected
in the usual way, with DAB, AEC etc.)
Tyramine-fluorochrome conjugates provide an even simpler
approach. They amount to methods for peroxidase activity
that generate fluorescent products that cannot be washed
off the tissue because they are covalently bound.
Tyramide methods is well suited to procedures with more
than one fluorescent label applied to the same section.
Several publications describe these techniques. Hopman
et al (1998) J Histochem Cytochem 46: 771-777 give
simple instructions for making your own conjugates of
tyramine with biotin, digoxigenin and various
fluorochromes. Similar instructions appeared also in the
earliest publications, which were for non-histological
applications (Bobrow et al., 1989 J Immunol Methods
125: 279-285, and others).
A few comments on the Histonet listserver in the last
year or so have indicated that the high sensitivity of
tyramide amplification methods can carry the price of
"excessive" staining (of everything). This should be
avoidable by diluting the reagents or by using a less
sensitive method for peroxidase activity.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1